This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Enveloped viruses have multiple mechanisms by which they inhibit infection of a target cell by more than one virion. Here we investigate the ability of influenza A virus hemagglutinin (HA), neuraminidase (NA), and M2 protein pumps to block entry of HA-pseudotyped retroviruses and infectious H1N1 and H3N2 influenza A viruses. We observed that NA from multiple isolates, but not HA or M2 or an enzymatically inactive form of NA, inhibited viral superinfection. Superinfection was markedly increased in the presence of the neuraminidase inhibitors oseltamivir carboxylate and zanamivir. These inhibitors also prevented removal of alpha-2,3- and alpha-2,6-linked sialic acid (SA) observed in cells expressing NA or infected with influenza A viruses. Our data show that the efficiency of SA digestion by NA can regulate viral superinfection, and suggest a functional role for cell-associated NA. Using these studies as a basis we established an RNAi-screen for host proteins that modulate influenza A virus replication. This screen identified IFITM3 has a major restriction factor regulating influenza A virus entry.